The present invention relates to novel galanin receptor proteins and partial peptides thereof; novel DNAs containing a galanin receptor protein or partial peptide-encoding DNA; processes for producing said galanin receptor protein (or partial peptide); use of said receptor protein (or partial peptide) and said protein (or partial peptide)-encoding DNA; a method of measuring the physiological actions of galanin using a galanin receptor protein-expressing cell or the galanin receptor protein; a method of screening galanin receptor agonists/antagonists using the galanin receptor protein-expressing cell or galanin receptor protein; a kit for said screening; an agonist or antagonist obtained by said screening method; and a pharmaceutical composition containing said agonist or antagonist.
The present invention also relates to a novel mouse pancreatic xcex2 cell line MIN6-derived galanin receptor protein and a partial peptide thereof; a novel DNA coding for said mouse galanin receptor protein or its partial peptide; processes for producing said mouse galanin receptor protein or its partial peptide; use of said mouse galanin receptor protein and said protein or peptide-encoding DNA; a method of measuring the physiological actions of galanin using a mouse-derived cell line MIN6 or the mouse galanin receptor protein; and a method of screening a galanin receptor agonist/antagonist using said mouse-derived cell line MIN6 or the receptor protein.
The present invention also relates to a novel human galanin receptor protein; a partial peptide of the human galanin receptor protein; a novel DNA which codes for the galanin receptor protein or partial peptide; a vector carrying said DNA; a transformant harboring said vector; a process for producing the human galanin receptor protein (or its partial peptide); a method of screening a galanin receptor agonist/antagonist using the human galanin receptor protein or a human galanin receptor protein-expressing cell (including the transformant); a kit for said screening; an agonist or an antagonist, obtained by said screening method; and a pharmaceutical composition containing said agonist or antagonist.
A variety of hormones, neurotransmitters and the like control, regulate or adjust the functions of living bodies via specific receptors located in cell membranes. Many of these receptors mediate the transmission of intracellular signals via activation of a guanine nucleotide-binding protein (hereinafter, sometimes referred to as xe2x80x9cG proteinxe2x80x9d) with which the receptor is coupled and possess the common (homologous) structure, i.e. seven transmembranes (membrane-spanning regions (domains)). Therefore, such a receptor is generically referred to as xe2x80x9cG protein coupled receptorxe2x80x9d or xe2x80x9cseven transmembrane (membrane-spanning) receptorxe2x80x9d.
G protein coupled receptor proteins which are widely distributed in the functional cellular surface of cells and organs in the living bodies have a very important role as targets for molecules such as hormones, neurotransmitters and physiologically active substances, which molecules control, regulate or adjust the functions of living bodies.
The pancreas plays an important role of carrying out the carbohydrate metabolism by secreting not only a digestive fluid but also glucagon and insulin. Insulin is secreted from the xcex2 cells and its secretion is promoted chiefly by glucose. It has been known that a variety of receptors exist in the xcex2 cells, and the secretion of insulin is controlled by various factors such as peptide hormones (galanin, somatostatin, gastric inhibitory polypeptide, glucagon, amylin, etc.), sugars (mannose, etc.), amino acids, and neurotransmitters in addition to glucose. As for the galanin and amylin, however, there has not yet been reported any discovery concerning the structure of their receptor protein cDNA. It is not known whether there exist any unknown receptor proteins or receptor protein subtypes.
It is a very important means in investigating development of new pharmaceuticals to clarify the relation between substances controlling the complicated functions of pancreas and specific receptors thereto. In order to develop new pharmaceuticals by conducting an effective screening of agonists and antagonists to the receptor proteins for controlling the functions of pancreas, it was necessary to investigate the function of receptor protein genes and also to express them in a suitable expression system.
By utilizing the fact that a G protein coupled receptor protein exhibits homology in part of the structure thereof at the amino acid sequence level, an experiment of looking at DNAs coding for novel receptor proteins relying upon a polymerase chain reaction (hereinafter simply referred to as xe2x80x9cPCRxe2x80x9d) has recently been made.
Galanin is a peptide existing in central and peripheral areas and, in central area, it shows an action of inhibition of liberation of neurotransmitter (acetylcholine) (European Journal of Pharmacology, vol.164, 355-360, 1989) and an action of antagonizing foreign acetylcholine (Proceedings of National Academy of Sciences, U.S.A., vol.85, 9841-9845, 1988) while, in pancreas, it shows a pharmacological action such as inhibition of insulin secretion (Diabates, vol. 34, 192-196, 1985). It has been also confirmed that galanin has an effect of inhibiting the behavior of learning (Neuroscience Letters, vol.88, 331-335, 1988) and of inhibiting the feeling of fullness after a meal. Such findings suggest a possibility that, if pharmaceuticals which inhibit the action of galanin are developed, they may be used as intelligence tropic agents and as remedies for obesity and for diabetes.
All of the pharmacological actions of galanin take place via a specific galanin receptor existing in target tissues. Accordingly, the simplest means for inhibiting the action of galanin is to develop pharmaceuticals which specifically inhibit the reaction of galanin with the receptor, i.e. galanin receptor antagonists. In the development of galanin receptor antagonists, it is usually necessary to conduct a receptor binding experiment. In the case of galanin, experiments on galanin receptor binding using membrane fractions of brain hippocampal formation (European Journal of Biochemistry, vol. 181, 269-276, 1989) and of stomach and duodenum (Peptides, vol. 11, 333-338, 1990) have been reported already.
It has been also reported that there is a specific galanin receptor in Rin-m-5F cells obtained from rat pancreas (Endocrinology, vol. 124, 2635-2641, 1989). According to the above-mentioned reports, it is already possible to conduct a galanin receptor binding experiment. However, the amount of the galanin receptor in those membrane fractions is as low as around 50 fmol/mg and, therefore, it was necessary to use a large amount of cell fractions for one measurement.
Galanin exhibits the above-mentioned pharmacological actions in living body and, if the actions can be easily measured in vitro, that will be meaningful for the process of developing the receptor antagonists. It has been reported already that the action of inhibiting the insulin secretion by galanin can be substituted with an in vitro measurement using Langerhans islet isolated from pancreas (European Journal of Pharmacology, Vol. 203, 111-114, 1991). However, Langerhans island is required to be isolated upon each experiment and, therefore, this method is not easily accomplished.
As easier means, several methods using pancreatic xcex2-cell strains (Rin-m-5F cells) have reported. They are, for example, a method in which an effect of galanin receptor to a second messenger system (i.e. an activity of inhibiting the adenylate cyclase) is measured (European Journal of Biochemistry, Vol. 177, 147-152, 1988) and a method in which an activity of opening the potassium channel is measured (Proceedings of National Academy of Sciences, U.S.A., Vol. 85, 1312-1316, 1988). A method in which the activity of inhibiting the insulin secretion of galanin using said cell strain has been reported too. However, those methods are applicable only for insulin secretion which is dependent upon forskolin (an adenylate cyclase activator) and the measurement for secretion of glucose-dependent insulin is not possible. Further, the secretion amount of insulin is small and the sensitivity is low.
After those, a method of preparing the xcex2-cell strains using pancreas of transgenic mice (Proceedings of National Academy of Sciences, U.S.A., Vol. 85, 9037-9041, 1988) has been developed and establishments of cell strains such as xcex2TC-1 cells (Proceedings of National Academy of Sciences, U.S.A., Vol. 85, 9037-9041, 1988), IgSV 195 cells (Diabates, Vol. 38, 1056-1062, 1989) and MIN6 cells (Endocrinology, Vol. 127, 126-132, 1990) have been reported. Among those, MIN6 cells hold the ability of insulin secretion depending upon the glucose concentration (which is a differentiating function inherent to xcex2-cells) in the best manner and, in addition, they secret insulin in a high amount. However, it has not been known yet that galanin receptor protein is expressed in said MIN6 cells. In addition, there has been no proposal yet for an evaluating system for the biological activity of galanin and also for an effective method for screening the galanin receptor agonist or antagonist using the MIN6 cells.
Recently, cDNA which codes for human galanin receptor protein was cloned and its nucleotide sequence and also its amino acid sequence encoded by said cDNA have been disclosed (Proceedings of National Academy of Sciences, U.S.A., Vol. 91, 9780-9783, Oct. 11, 1994). However, there is no disclosure at all for a specific means for screening the galanin receptor agonist/antagonist using said receptor.
Under such circumstances, a method for screening and assessing galanin receptor agonist/antagonist in an efficient manner is still desired.
Galanin is a polypeptide comprising 29 amino acid residues separated from porcine small intestine [Tatemoto, K. et al., FEBS Letter, 164, 124-128(1983)] and its primary structure is hardly similar to those of other brain and intestinal hormones. Galanin immunoactivity is widely distributed in central nervous system and peripheral nervous system together with its receptor [Scofitsch, G. and Jacobowitz, D. M., Peptides, 6, 509-546(1985); Melander, T. et al., Journal of Comparative Neurology, 248, 475-517(1986); Rokaeus, A., Trends in Neuroscience, 10,158-164(1987)] and, since its distribution pattern is identical with the region containing the traditional neurotransmitters such as 5-HT, noradrenaline and acetylcholine, it is likely that galanin is present together with such neurotransmitters and controls the prenervous and postnervous actions by those neurotransmitters.
Galanin has many physiological actions and, in central nervous system, it strongly inhibits the single synaptic reflection in spinal nerve [Yanagisawa, M. et al., Neuroscience Letter, 70, 278-282(1986)] and its action is known to be far stronger than somatostatin. In addition, the physiological importance of galanin in nerve center has been greatly suggested because of stimulation of action for taking food [Kyrokouli, S. E. et al., European Journal of Pharmacology, 122, 159-160(1986)], participation in formation of memory [Crawley, J. N. and Wenk, G. L., Trends in Neuroscience, 12,278-282(1989)], inhibition of dopamine in median elevation [Nordstrom, O. et al., Neuroscience Letter, 73, 21-26(1987)], inhibition of release of acetylcholine in hippocampal double sides [Fisone, G. et al., Proceedings of the National Academy of Sciences of U.S.A., 84, 7339-7343(1987)], a decrease in metabolic circulation of 5-HT [Fuxe K., et al., Acta. Physiol. Scand., 133, 579-581(1988)] and a decrease in a glutamic acid release by activation of ATP-sensitive K+ channel [Ben-Ari, Y., European Journal of Neuroscience, 2, 62-68(1990)] as a result of administration of galanin to paraventricular nucleus of rats.
Especially, galanin is an only neuropeptide in which choline acetyltransferase is coexisting in the medial septal nucleus, nucleus of diagonal band and basal nucleus [Melender, T. et al., Brain Research, 360, 130-138(1985); Melender, T. et al., Neuroscience Letter, 19, 223-240(1986); Chen-Palay, V., Brain Research Bulletin, 21, 465-472(1988)] and is known to act on cholinergic nerves in an inhibiting manner while, on the other hand, it is expected that, since denaturation in cholinergic nerves is noted in those sites in Alzheimer""s disease, galanin antagonist may prevent the denaturation of the cholinergic nerves in Alzheimer""s disease or the like [Whitehouse, P. J., et al., Science, 215, 1237-1239(1982); Chen-Palay, V., Journal of Comparative Neurology, 273,543-557 (1988)]. In hypophysis, action of stimulating the secretion of growth hormones and prolactin has been noted [Tanoh, T., et al., Neuroendocrinology, 54, 83-88(1991); Koshiyama, H., et al., Neuroscience Letter, 75, 49-54(1987)]. Particularly in the secretion of growth hormones, participation of cholinergic neuron via adjustment of secretion of hypothalamic somatostatin is noted.
On the other hand, in peripheral systems, galanin inhibits the basal secretion of insulin both in vivo and in vitro [McDnald, T. J. et al., Diabates, 34, 192-196(1985); Takeda, Y. et al., Biomedical Research, 8 (Suppl.), 117-125 (1987); Lindskog, S. et al., Acta. Physiol. Scand., 129, 305-309(1987)] and, in addition, it inhibits the release of insulin by stimulation of glucose [Dunning, B. E. and Taborsky, G. J., Jr., Diabates, 37, 1157-1162(1988)]. When further immunohistological observation that nerve fiber net containing a dense galanin immunoactivity is noted around Langerhans islet of xcex2 cells is taken into consideration, it has been strongly suggested that galanin is one of the nerve controlling factors for secretion of pancreatic hormones, especially insulin. It is also noted that, in stomach, galanin inhibits the basal secretion of somatostatin on a dose-depending manner or it inhibits the secretion of somatostatin or gastrin by stimulation of GRP and that nerve fiber net containing galanin immunoactivity is observed in stomach and, accordingly, it is suggested that, even in stomach, galanin acts as one of the important nerve controlling factors for adjusting the secretion in stomach [Yanaihara, N. et al., in xe2x80x9cGalaninxe2x80x9d (ed. by Hokfelt, T. et al.), Macmillan Press, 185-196(1991)].
From the above descriptions, it is understood that galanin agonist is useful as a pharmaceutical agent such as a stimulant for secretion of growth hormones and an inhibitor for secretion of insulin and that galanin antagonist is useful as another pharmaceutical agent such as an inhibitor for secretion of growth hormones and a stimulant for secretion of insulin.
Usually, in developing agonists and antagonists for physiologically-active substances, investigations are made on the compounds which have high affinity with the receptors to which said substance is specifically bonded. At present, bovine hippocampal membrane fraction is used as a galanin receptor but, because of the difference in the animal species used, there is no guarantee that the compound exhibiting a high affinity to said membrane fraction has a high affinity to human galanin receptor as well. Human galanin receptor cDNA has been cloned and reported to exhibit an expression in COS cells [Habert-Ortoll, E. et al., Proceedings of the National Academy of Sciences, U.S.A., 91, 9780-9783(1994)] but, since the expressed amount is small and the expression is mere transient, it is thought to be unsuitable for screening.
One object of the present invention is to provide novel galanin receptor proteins and partial peptides thereof or salts thereof; DNAs comprising a DNA coding for said galanin receptor protein or its partial peptide; vectors carrying said DNA; transformants harboring said vector; cell membrane fractions obtained from said transformant; processes for producing said receptor protein or its partial peptide, or a salt thereof; methods for measuring the physiological actions of galanin using the galanin receptor protein (including a cell membrane fraction containing the receptor protein) or a galanin receptor protein-expressing cell (including the transformant); screening methods for a galanin receptor agonist/antagonist using the galanin receptor protein or a galanin receptor protein-expressing cell (including the transformant); kits for said screening; agonists or antagonists, obtained by said screening method; pharmaceutical compositions containing said agonist or antagonist; antibodies against said receptor protein; immunoassays using said receptor protein or said antibody; and use of said receptor protein and encoding DNA.
Another object of the present invention is to provide novel mouse pancreatic xcex2 cell line MIN6-derived galanin receptor proteins or partial peptides thereof; DNAs comprising a DNA coding for said galanin receptor protein or partial peptide; processes for producing said receptor protein or its partial peptide; methods of measuring the physiological actions of galanin using a mouse-derived cell line MIN6 or the galanin receptor protein; screening methods for a galanin receptor agonist/antagonist using said mouse-derived cell line MIN6 or the receptor protein; antibodies against said receptor protein; immunoassays using said receptor protein or said antibody; and use of said galanin receptor protein or said receptor protein peptide-encoding DNA.
Human galanin receptor proteins manufactured by the conventional method and the COS cells which express said human galanin receptor protein are insufficient as receptor samples for conducting a screening for galanin receptor agonist/antagonist. Consequently, there has been a demand for developing a more practical method for manufacturing human galanin receptor proteins.
If it is possible to screen the agonist/antagonist of galanin receptor using human galanin receptor protein, it is now possible to overcome the disadvantage by the use of experimental animals (for example, the possibility that, due to a difference in species, compounds which do not achieve an effect to human being may be obtained) whereby it is expected to conduct a development of pharmaceutical agents effective to human being in an efficient manner.
Yet another object of the present invention is to provide novel human galanin receptor proteins; partial peptides of the human galanin receptor protein; novel DNAs which code for the galanin receptor protein or partial peptide; vectors carrying said DNA; transformants harboring said vector; cell membrane fractions obtained from said transformant; processes for producing the human galanin receptor protein (or its partial peptide); methods for measuring the physiological actions of galanin using a human galanin receptor protein-expressing cell, said human galanin receptor protein; screening methods for a galanin receptor agonist/antagonist using a human galanin receptor protein-expressing cell (including the transformant); kits for said screening; agonists or antagonists, obtained by said screening method; pharmaceutical compositions containing said agonist or antagonist; antibodies against said human galanin receptor protein; immunoassays using said receptor protein or said antibody; and use of said human galanin receptor protein and encoding DNA.
In order to achieve the above-mentioned aims, the present inventors have made extensive investigations. As a result, the present inventors have succeeded in synthesizing DNA primers effective in efficiently isolating DNAs (DNA fragments) coding for G protein coupled receptor proteins by PCR techniques. The present inventors have succeeded in amplifying cDNA derived from various cells with said synthetic DNA primer, and have forwarded the analysis. Thus, the present inventors have succeeded in isolating novel G protein coupled receptor protein-encoding cDNAs, in determining the partial structure thereof, and have considered that the isolated cDNAs are homologous to known G protein coupled receptors at the nucleotide sequence level and at the amino acid sequence level and are each coding for a novel galanin receptor protein. Based upon the above knowledge, the present inventors have discovered that these DNAs make it possible to obtain a cDNA having a full length open reading frame (ORF) of the receptor protein, hence, to produce the receptor protein. The inventors have further succeeded in sequencing an entire amino acid sequence and entire nucleotide sequence of said galanin receptor protein.
The present inventors have found that, when said receptor protein expressed by a suitable means is used, an agonist or an antagonist to said receptor protein can be screened in vivo or from natural or nonnatural compounds by a receptor protein binding experiment or by a measurement of intracellular second messenger as an index. The present inventors have further found that said agonist and antagonist can be developed as preventive and therapeutic agents for the diseases or symptoms related to or caused by galanin.
The present inventors have furthermore found that the glucose or forskolin-dependent insulin secretion in cells expressing said galanin receptor protein is inhibited by galanin. That has been a finding for the first time. Depending upon said finding, the present inventors have found an easy and simple method for measuring the activity of galanin and galanin antagonist. At the same time, the present inventors have also found that the cell membrane fractions of cells expressing said galanin receptor protein contain large amount of galanin receptors and succeeded in establishing a screening for galanin receptor agonist/antagonist using the cell membrane fractions thereof.
For example, the present inventors have amplified G protein coupled receptor protein-encoding cDNA derived from mouse pancreatic xcex2-cell strain MIN6 using a synthetic DNA primer for more effective isolation thereof, whereby its analysis has been carried out.
As a result thereof, the present inventors have succeeded in isolating the mouse-derived cDNA fragment which codes for a novel G protein coupled receptor protein and in elucidating its partial structure. In said mouse-derived G protein coupled receptor protein, there are similarities (homologies) at DNA and amino acid levels to the known G protein coupled receptor and, therefore, it is believed that it codes for a novel receptor protein exhibiting an expressing function in mouse pancreas.
The present inventors further continued their studies and have succeeded in cloning cDNA having a full-length translation unit and in analyzing an entire amino acid sequence and an entire nucleotide sequence of said receptor protein. Since said mouse-derived G protein coupled receptor protein has a high homology at DNA and amino acid levels to the human-derived galanin receptor protein (Proceedings of National Academy of Sciences, U.S.A., 91, 9780-9783, 1994), it has been found that said mouse-derived G protein coupled receptor protein is identical with a mouse-derived galanin receptor protein.
Furthermore, the present inventors have newly found that the glucose or forskolin-dependent insulin secretion of MIN6 cells is inhibited by galanin. Based upon said finding, the present inventors have found an easy and simple method for measuring the activity of galanin and galanin antagonist. At the same time, the present inventors have also found that the cell membrane fractions of MIN6 cells contain large amount (0.5-1.0 pmol/mg) of galanin receptor and succeeded in establishing a method of screening galanin receptor agonist/antagonist using the cell membrane fractions of MIN6 cells.
To be more specific, the present inventors have amplified and cloned novel cDNA fragments derived from mouse pancreatic xcex2-cell strain MIN6 as shown in FIG. 1 by PCR and, from the result of analysis of their sequence, have clarified that they code for a novel receptor protein. When said sequence was translated into amino acid sequences, third, fourth, fifth and sixth transmembrane domains were confirmed on hydrophobic plots (FIG. 2). The size of the amplified DNA was about 400 bp which was almost same as that of the known G protein coupled receptor protein.
The inventors have retrieved the data base based on, as a template, the nucleotide sequence of the isolated DNA and observed 36% homology to human-derived somatostatin receptor subtype 4 (JN0605), 30% homology to human-derived somatostatin receptor subtype 2 (B41795), and 30% homology to rat-derived ligand unknown receptor (A39297), respectively (FIG. 3), which are known G protein coupled receptor proteins. The aforementioned abbreviations in parentheses are reference numbers that are assigned when they are registered as data to NBRF-PIR/Swiss-PROT and are, usually, each called xe2x80x9cAccession Numberxe2x80x9d or xe2x80x9cEntry Namexe2x80x9d.
Moreover, the present inventors have prepared cDNA from the poly(A)+RNA fractions extracted from MIN6 cells and have inserted said cDNA into lambda gt22 phage to prepare a cDNA library. Further, the present inventors have screened the cDNA library using, as a probe, the G protein coupled receptor protein cDNA fragment p3H2-34 obtained by PCR and succeeded in cloning cDNA which completely codes for the G protein coupled receptor protein of the present invention. A nucleotide sequence of said cDNA and an amino acid sequence encoded thereby are given in FIG. 4. A hydrophobic plotting was conducted based upon said amino acid sequence and the first, second, third, fourth, fifth, sixth and seventh transmembrane domains were confirmed (FIG. 5). The G protein coupled receptor protein of the present invention has 92% homology at the amino acid level to the known human galanin receptor protein.
In another aspect, the present inventors have succeeded, for example, in cloning a DNA which codes for novel human galanin receptor protein having an amino acid sequence which differs from that of known human galanin receptor protein. In the known human galanin receptor protein, the fifteenth amino acid in its amino acid sequence is Cys while, in the human galanin receptor protein of the present invention, the fifteenth amino acid in its amino acid sequence (SEQ ID NO: 5 and FIGS. 12 and 13) is Trp. In addition, in the nucleotide sequence of DNA which codes for the known human galanin receptor protein, the base sequence which codes for the fifteenth amino acid of said human galanin receptor protein is 15Cys (TGT) while, in the base sequence of DNA which codes for the human galanin receptor protein of the present invention, the base sequence which codes for the fifteenth amino acid in said human galanin receptor protein is 15Trp (TGG).
The present inventors have further succeeded in manufacturing a CHO cell strain which expresses far more amount of the human galanin receptor protein of the present invention than the COS cells which express the known human galanin receptor protein [Habert-Ortoll, E. et al., Proceedings of the National Academy of Sciences of the U.S.A., 91, 9780-9783 (1994)]. It has been furthermore found that, when said CHO cell strain of the human galanin receptor protein of the present invention or partial peptide thereof is used, it is now possible to screen the human galanin receptor agonist/antagonist in an effective and reliable manner. Based upon those findings, the present inventors have continued various investigations and, as a result, they have achieved the present invention.
Accordingly, one aspect of the present invention is
(1) a galanin receptor protein comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5 and substantial equivalents thereto, or a salt thereof;
(2) the receptor protein according to the above (1), which is produced by a transformant CHO cell;
(3) a DNA which comprises a nucleotide sequence coding for a galanin receptor protein of the above (1);
(4) a vector comprising the DNA according to the above (3);
(5) a transformant carrying the vector according to the above (4);
(6) the transformant according to the above (5), wherein the host cell is a CHO cell;
(7) a process for producing a galanin receptor protein according to the above (1), which comprises culturing a transformant of the above (5) under conditions suitable to express said galanin receptor protein;
(8) a screening method for an agonist or antagonist of a galanin receptor protein according to the above (1), which comprises carrying out a comparison between:
(i) at least one case where galanin is contacted with at least one component selected from the group consisting of a galanin receptor protein according to the above (1), a partial peptide thereof and a mixture thereof, and
(ii) at least one case where galanin together with a compound to be tested is contacted with at least one component selected from the group consisting of a galanin receptor protein according to the above (1), a partial peptide thereof and a mixture thereof;
(9) a kit for the screening of one or more agonists or antagonists to a galanin receptor protein according to the above (1), which comprises at least one component selected from the group consisting of a galanin receptor protein according to the above (1), a partial peptide thereof and a mixture thereof; and
(10) an agonist or antagonist of a galanin receptor, which is obtained by the screening method according to the above (8) or by the kit according to the above (9).
Another aspect of the present invention is
(11) a mouse-derived galanin receptor protein comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 1 and substantial equivalents thereto; or a salt thereof;
(12) a mouse-derived galanin receptor protein according to the above (11), which comprises an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 2 and substantial equivalents thereto; or a salt thereof;
(13) a human galanin receptor protein comprising an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 5 and substantial equivalents thereto; or a salt thereof;
(14) a partial peptide of a galanin receptor protein according to the above (1), or a salt thereof;
(15) a partial peptide of a mouse-derived galanin receptor protein according to the above (11) or (12), or a salt thereof;
(16) a partial peptide of a human galanin receptor protein according to the above (13), or a salt thereof;
(17) a DNA which comprises a nucleotide sequence coding for a mouse-derived galanin receptor protein of the above (11) or (12);
(18) a DNA which comprises a nucleotide sequence coding for a human galanin receptor protein of the above (13);
(19) a DNA of the above (17) comprising a nucleotide sequence represented by SEQ ID NO: 3;
(20) a DNA of the above (17) comprising a nucleotide sequence represented by SEQ ID NO: 4;
(21) a DNA of the above (18) comprising a nucleotide sequence represented by SEQ ID NO: 6;
(22) a vector comprising a DNA according to the above (17);
(23) a vector comprising a DNA according to the above (18);
(24) a transformant (including a transfectant) carrying a vector of the above (22);
(25) a transformant (including a transfectant) carrying a vector of the above (23);
(26) a process for producing a mouse-derived galanin receptor protein or a salt thereof according to the above (11), which comprises culturing a transformant of the above (24) to produce said galanin receptor on the membrane of the transformant;
(27) a process for producing a human galanin receptor protein or a salt thereof according to the above (13), which comprises culturing a transformant of the above (25) under conditions to express said galanin receptor;
(28) a cell or membrane fraction containing a galanin receptor protein according to the above (1);
(29) a cell or membrane fraction containing a mouse-derived galanin receptor protein according to the above (11) or (12);
(30) a cell or membrane fraction containing a human galanin receptor protein according to the above (13);
(31) a screening method for a galanin receptor agonist and/or antagonist, which comprises using a galanin receptor protein according to the above (1), a partial peptide according to the above (14) or a cell or membrane fraction according to the above (28);
(32) a screening method for a mouse-derived galanin receptor agonist and/or antagonist, which comprises using a mouse-derived galanin receptor protein according to the above (11) or (12), a partial peptide according to the above (15) or a cell or membrane fraction according to the above (29);
(33) a screening method for a human galanin receptor agonist and/or antagonist, which comprises using a human galanin receptor protein according to the above (13), a partial peptide according to the above (16) or a cell or membrane fraction according to the above (30);
(34) a screening method for a galanin receptor agonist and/or antagonist, which comprises carrying out a comparison between:
(i) at least one case where galanin is contacted with at least one component selected from the group consisting of a galanin receptor protein or a salt thereof according to the above (1), a partial peptide or a salt thereof according to the above (14), a cell or membrane fraction according to the above (28), and a mixture thereof, and
(ii) at least one case where galanin together with a sample (including a compound) to be tested is contacted with at least one component selected from the group consisting of a galanin receptor protein or a salt thereof according to the above (1), a partial peptide or a salt thereof according to the above (14), a cell or membrane fraction according to the above (28), and a mixture thereof;
(35) a screening method for a mouse-derived galanin receptor agonist and/or antagonist, which comprises carrying out a comparison between:
(i) at least one case where galanin is contacted with at least one component selected from the group consisting of a mouse-derived galanin receptor protein or a salt thereof according to the above (11), a partial peptide or a salt thereof according to the above (15), and a mixture thereof, and
(ii) at least one case where galanin together with a sample (including a compound) to be tested is contacted with at least one component selected from the group consisting of a mouse-derived galanin receptor protein or a salt thereof according to the above (11), a partial peptide or a salt thereof according to the above (15), and a mixture thereof;
(36) a screening method for a human galanin receptor agonist and/or antagonist, which comprises carrying out a comparison between:
(i) at least one case where galanin is contacted with at least one component selected from the group consisting of a human galanin receptor protein or a salt thereof according to the above (13), a partial peptide or a salt thereof according to the above (16), a cell or membrane fraction according to the above (30), and a mixture thereof, and
(ii) at least one case where galanin together with a sample (including a compound) to be tested is contacted with at least one component selected from the group consisting of a human galanin receptor protein or a salt thereof according to the above (13), a partial peptide or a salt thereof according to the above (16), a cell or membrane fraction according to the above (30), and a mixture thereof;
(37) a kit for the screening of a galanin receptor agonist and/or antagonist, which comprises at least one component selected from the group consisting of a galanin receptor protein or a salt thereof according to the above (1), a partial peptide or a salt thereof according to the above (14), a cell or membrane fraction according to the above (28), and a mixture thereof;
(38) a kit for the screening of a mouse-derived galanin receptor agonist and/or antagonist, which comprises at least one component selected from the group consisting of a mouse-derived galanin receptor protein or a salt thereof according to the above (11) or (12), a partial peptide or a salt thereof according to the above (15), a cell or membrane fraction according to the above (29), and a mixture thereof;
(39) a kit for the screening of a human galanin receptor agonist and/or antagonist, which comprises at least one component selected from the group consisting of a galanin receptor protein or a salt thereof according to the above (13), a partial peptide or a salt thereof according to the above (16), a cell or membrane fraction according to the above (30), and a mixture thereof;
(40) a galanin receptor agonist and/or antagonist, obtained by a method according to any of the above (31) to (36) or a kit according to any of the above (37) to (39);
(41) a galanin receptor agonist and/or antagonist, obtained by a method according to the above (32) or (35) or a kit according to the above (38);
(42) a galanin receptor agonist and/or antagonist, obtained by a method according to the above (33) or (36) or a kit according to the above (39);
(43) a pharmaceutical composition comprising an effective amount of the galanin receptor agonist according to (40);
(44) a pharmaceutical composition comprising an effective amount of the galanin receptor agonist according to (41);
(45) a pharmaceutical composition comprising an effective amount of the galanin receptor agonist according to (42);
(46) a pharmaceutical composition comprising an effective amount of the galanin receptor antagonist according to (40);
(47) a pharmaceutical composition comprising an effective amount of the galanin receptor antagonist according to (41);
(48) a pharmaceutical composition comprising an effective amount of the galanin receptor antagonist according to (42);
(49) a pharmaceutical composition according to (43) which is an inhibitor for acetylcholine liberation, an inhibitor for insulin secretion, a stimulant for growth hormone secretion, an inhibitor for learning behavior or satiety;
(50) a pharmaceutical composition according to (46) which is an agent for promoting the acetylcholine liberation, an agent for inhibiting the growth hormone secretion, an agent for promoting the insulin secretion, an agent for promoting the learning behavior or an agent for promoting satiety;
(51) an antibody against at least one component selected from the group consisting of a galanin receptor protein or a salt thereof according to the above (1) and a partial peptide or a salt thereof according to the above (14);
(52) an antibody against at least one component selected from the group consisting of a mouse-derived galanin receptor protein or a salt thereof according to the above (11) or (12) and a partial peptide or a salt thereof according to the above (15); and
(53) an antibody against at least one component selected from the group consisting of a human galanin receptor protein or a salt thereof according to the above (13) and a partial peptide or a salt thereof according to the above (16).
To be more specific, the present invention relates to the following:
(54) a method of screening a galanin receptor agonist or antagonist, characterized in that, the binding amount of the labeled galanin with the galanin receptor protein or its salt according to (1) (e.g., the mouse-derived galanin receptor protein or its salt according to (11), etc.) or with the partial peptide or its salt according to (14) (e.g., the partial peptide of the mouse-derived galanin receptor protein or its salt according to (15), etc.) is measured in the case where the labeled galanin is contacted with the galanin receptor protein or its salt according to (1) (e.g., the mouse-derived galanin receptor protein or its salt according to (11), etc.) or with the partial peptide or its salt according to (14) (e.g., the partial peptide of the mouse-derived galanin receptor protein or its salt according to (15), etc.) and also in the case where the labeled galanin and the test compound are contacted with the galanin receptor protein or its salt according to (1) (e.g., the mouse-derived galanin receptor protein or its salt according to (11), etc.) or with the partial peptide or its salt according to (14) (e.g., the partial peptide of the mouse-derived galanin receptor protein or its salt according to (15), etc.) and the comparison is made between them;
(55) a method of screening a galanin receptor agonist or antagonist, characterized in that, the labeled galanin is contacted with the cells (except mouse-derived MIN6 cells [FERM BP-4954]) containing the galanin receptor protein according to (1) (e.g., the mouse-derived galanin receptor protein according to (11), etc.) and the labeled galanin and the test compound are contacted with the cells (except mouse-derived MIN6 cells [FERM BP-4954]) containing the galanin receptor protein according to (1) (e.g., the mouse-derived galanin receptor protein according to (11), etc.) and the binding amounts of the labeled galanin with said cells in both cases are measured and compared;
(56) a method of screening a galanin receptor agonist or antagonist, characterized in that, the labeled galanin is contacted with the cell membrane fractions of cells (except mouse-derived MIN6 cells [FERM BP-4954]) containing the galanin receptor protein according to (1) (e.g., the mouse-derived galanin receptor protein according to (11), etc.) and the labeled galanin and the test compound are contacted with the cell membrane fraction of cells (except mouse-derived MIN6 cells [FERM BP-4954]) containing the galanin receptor protein according to (1) (e.g., the mouse-derived galanin receptor protein according to (11), etc.) and the binding amounts of the labeled galanin with the membrane fractions of said cells in both cases are measured and compared;
(57) a method of screening a galanin receptor agonist or antagonist, characterized in that, the labeled galanin is contacted with the galanin receptor protein according to (1) (e.g., the mouse-derived galanin receptor protein according to (11), etc.) expressed in cell membranes of the transformant according to (5) (e.g., the mouse-derived galanin receptor protein-expressible transformant according to (24), etc.) by culturing said transformant and the labeled galanin and the test compound are contacted with the galanin receptor protein according to (1) (e.g., the mouse-derived galanin receptor protein according to (11), etc.) expressed in cell membranes of the transformant according to (5) (e.g., the mouse-derived galanin receptor protein-expressible transformant according to (24), etc.) by culturing said transformant and the binding amounts of the labeled galanin with said galanin receptor in both cases are measured and compared;
(58) a method of screening a galanin receptor agonist or antagonist, characterized in that, galanin is contacted with the cells (except the mouse-derived MIN6 cells [FERM BP-4954]) containing the galanin receptor protein according to (1) (e.g., the mouse-derived galanin receptor protein according to (11), etc.) and galanin and the test compound are contacted with the cells (except the mouse-derived MIN6 cells [FERM BP-4954]) containing the galanin receptor protein according to (1) (e.g., the mouse-derived galanin receptor protein according to (11), etc.) and the resulting cell-stimulating activities via the galanin receptor protein in both cases are measured and compared;
(59) a method of screening a galanin receptor agonist or antagonist, characterized in that, galanin is contacted with the galanin receptor protein according to (1) (e.g., the mouse-derived galanin receptor protein according to (11), etc.) expressed in cell membranes of the transformant according to (5) (e.g., the mouse-derived galanin receptor protein-expressible transformant according to (24), etc.) by culturing said transformant and galanin and the test compound are contacted with the galanin receptor protein according to (1) (e.g., the mouse-derived galanin receptor protein according to (11), etc.) expressed in cell membranes of the transformant according to (5) (e.g., the mouse-derived galanin receptor protein-expressible transformant according to (24), etc.) by culturing said transformant and the resulting cell stimulating activities via the galanin receptor protein are measured and compared;
(60) a method of screening according to (58) or (59) in which the cell-stimulating activity is an activity which accelerates or inhibits arachidonic acid liberation, acetylcholine liberation, intracellular Ca2+ liberation, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential variation, phosphorylation of intracellular protein, activation of c-fos, a decrease in pH, insulin secretion, etc. (especially the activity which accelerates or inhibits the intracellular cAMP production or insulin secretion);
(61) a galanin receptor agonist or antagonist obtained by a screening methods according to any of (31), (34) (e.g., (32), (35), etc.) and (54) to (60);
(62) an agent for inhibiting acetylcholine liberation, insulin secretion, learning behavior or feeling of satiety after a meal characterized in containing the galanin receptor agonist according to (61);
(63) an agent for accelerating acetylcholine liberation, insulin secretion, behavior of learning or feeling of fulfillment after a meal characterized in containing the galanin receptor antagonist according to (61);
(64) an intelligence tropic agent or a remedy for obesity or for diabetes characterized in containing the galanin receptor antagonist according to (40) (e.g., (41), etc.) or (61);
(65) a kit for screening according to (37) (e.g., (38), etc.), characterized in comprising a cell containing the galanin receptor protein according to (1) (e.g., the mouse-derived galanin receptor protein according to (11), etc.);
(66) a kit for screening according to (37) (e.g., (38), etc.), characterized in containing the membrane fractions of the cells which contain the galanin receptor protein according to (1) (e.g., the mouse-derived galanin receptor protein according to (11), etc.);
(67) a galanin receptor agonist or antagonist obtained by the use of the kit for screening according to (37) (e.g., (38), etc.), (65) or (66);
(68) an agent for inhibiting acetylcholine liberation, insulin secretion, learning behavior or feeling of fulfillment after a meal characterized in containing the galanin receptor agonist according to (67);
(69) an agent for accelerating acetylcholine liberation, insulin secretion, behavior of learning or feeling of fulfillment after a meal characterized in containing the galanin receptor antagonist according to (67);
(70) an intelligence tropic agent or a remedy for obesity or for diabetes characterized in containing the galanin receptor antagonist according to (40) (e.g., (41), etc.) or (69); and
(71) a method of quantitative determination of the galanin receptor protein or its salt according to (1) (e.g., the mouse-derived galanin receptor protein or its salt according to (11), etc.) or the partial peptide or its salt according to (14) (e.g., the partial peptide of the mouse-derived galanin receptor protein or its salt according to (15), etc.), characterized in that, the antibody according to (51) (e.g., the antibody according to (52), etc.) is contacted with the galanin receptor protein or its salt according to (1) (e.g., the mouse-derived galanin receptor protein or its salt according to (11), etc.) or the partial peptide or its salt according to (14) (e.g., the partial peptide of the mouse-derived galanin receptor protein or its salt according to (15), etc.).
The present invention furthermore provides the following:
(72) a method of measuring the physiological activity of galanin, characterized in that, the biological activity of the mouse-derived MIN6 cells when the mouse-derived MIN cells (FERM BP-4954) or the cell membrane fractions thereof are contacted with galanin;
(73) a method of screening a galanin receptor agonist or antagonist, characterized in that, a comparison is made between the cases where (i) galanin is contacted with the mouse-derived MIN6 cells (FERM BP-4954) or cell membrane fractions thereof and (ii) galanin and the test compound are contacted with the mouse-derived MIN6 cells (FERM BP-4954) or cell membrane fractions thereof;
(74) a kit for screening for a galanin receptor agonist or antagonist characterized in containing the mouse-derived MIN6 cells (FERM BP-4954) or cell membrane fractions thereof;
(75) a galanin receptor agonist or antagonist obtained by the method for screening according to (73) or by the kit for screening according to (74);
(76) an inhibitor for liberation of acetylcholine, an inhibitor for secretion of insulin, an inhibitor for the behavior of learning or an inhibitor for feeling satiety after a meal characterized in containing the galanin receptor agonist according to (75);
(77) an accelerator for liberation of acetylcholine, an accelerator for secretion of insulin, an accelerator for the behavior of learning or an accelerator for feeling satiety after a meal characterized in containing the galanin receptor antagonist according to (75);
(78) a method for screening a galanin receptor agonist or antagonist, characterized in that, the labeled galanin is contacted with the mouse-derived MIN6 cells (FERM BP-4954) and the labeled galanin and the test compound are contacted with the mouse-derived MIN6 cells (FERM BP-4954) and the binding amounts of the labeled galanin with said mouse-derived galanin MIN6 cells in both cases are measured and compared;
(79) a method of screening a galanin receptor agonist or antagonist, characterized in that, the labeled galanin is contacted with the cell membrane fractions of the mouse-derived MIN6 cells (FERM BP-4954) and the labeled galanin and the test compound are contacted with the cell membrane fractions of the mouse-derived MIN6 cells (FERM BP-4954) and the binding amounts of the labeled galanin with said membrane fractions of the mouse-derived MIN6 cells in both cases are measured and compared;
(80) a method of screening a galanin receptor agonist or antagonist, characterized in that, galanin is contacted with the mouse-derived MIN6 cells (FERM BP-4954) and galanin and the test compound are contacted with the mouse-derived MIN6 cells (FERM BP-4954) and the resulting cell-stimulating activities via the mouse-derived galanin receptor (especially the activity of secretion of insulin from MIN6 cells or the activity of inhibiting or accelerating the cAMP production in the MIN6 cells) in both cases are measured and compared;
(81) a method of screening according to the above (80) in which the cell-stimulating activity is an activity for accelerating or inhibiting the arachidonic acid liberation, acetylcholine liberation, intracellular Ca2+ liberation, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential variation, phosphorylation of intracellular protein, activation of c-fos, a decrease in pH, secretion of insulin, etc. (especially the activity which accelerates or inhibits the intracellular cAMP production or the insulin secretion);
(82) a galanin receptor agonist or antagonist obtained by a method of screening according to any of (73) and (78) to (81);
(83) an inhibitor for liberation of acetylcholine, an inhibitor for secretion of insulin, an inhibitor for the behavior of learning and an inhibitor for feeling fulfillment after a meal characterized in containing the galanin receptor agonist according to (75) or (82);
(84) an accelerator for liberation of acetylcholine, an accelerator for secretion of insulin, an accelerator for the behavior of learning and an accelerator for feeling satiety after a meal characterized in containing the galanin receptor antagonist according to (75) or (82); and
(85) an intelligence tropic agent or a remedy for obesity or for diabetes characterized in containing the galanin receptor antagonist according to (75) or (82).
Yet another aspect of the present invention is:
(86) a partial peptide according to (16) in which the partial peptide is a region exposed outside the cell membrane of the human galanin receptor protein molecule according to (13);
(87) a vector according to (23) in which the vector is an expression vector for the human galanin receptor protein as indicated by pTS863;
(88) a transformant according to (25) in which the host cell is a CHO cell;
(89) a CHO cell according to (88) in which the CHO cell is CHO/pTS863-5 or CHO/pTS863-7;
(90) a cell or cell membrane fraction thereof according to (30) in which the cell is CHO/pTS863-5 or CHO/pTS863-7;
(91) a method of screening the galanin receptor agonist or antagonist according to (33), which comprises carrying out a comparison between the cases where (i) galanin is contacted with the human galanin receptor protein or salt thereof according to (13) or with the partial peptide or salt thereof according to (16) and (ii) galanin and the test compound are contacted with the human galanin receptor protein or salt thereof according to (13) or with the partial peptide or salt thereof according to (16);
(92) a method of screening the galanin receptor agonist or antagonist according to (33), which comprises measuring and comparing the binding amounts of the labeled galanin to said human galanin receptor protein, partial peptide thereof or salt thereof in the cases where (i) the labeled galanin is contacted with the human receptor protein or salt thereof according to (13) or with the partial peptide or salt thereof according to (16) and (ii) the labeled galanin and the test compound are contacted with the human galanin receptor protein or salt thereof according to (13) or with the partial peptide or salt thereof according to (16);
(93) a method of screening the galanin receptor agonist or antagonist according to (33), which comprises carrying out a comparison between the cases where (i) the labeled galanin is contacted with the cell or cell membrane fraction thereof according to (30) and (ii) the labeled galanin and the test compound are contacted with the cell or the cell membrane fraction thereof according to (30);
(94) a method of screening the galanin receptor agonist or antagonist according to (33), which comprises measuring and comparing the binding amounts of the labeled galanin with said cell or cell membrane fraction thereof in the cases where (i) the labeled galanin is contacted with the cell or the cell membrane fraction thereof according to (30) and (ii) the labeled galanin and the test compound are contact with the cell or the cell membrane fraction thereof according to (30);
(95) a method of screening the galanin receptor agonist or antagonist according to (33), which comprises measuring and comparing cell stimulating activities via the recombinant human galanin receptor (for example, activities which promote or inhibit the opening of K+ channel, closing of N type Ca+ channel, liberation of arachidonic acid, liberation of acetylcholine, variations in intracellular Ca2+ concentration, inhibition of intracellular cAMP production, production of inositol phosphate, cell membrane potential changes, phosphorylation of intracellular protein, activation of c-fos, decrease in pH, cell migration activity, secretion of hormones, activation of G protein and cell promulgation, etc.) in the cases where (i) galanin is contacted with the cell or the cell membrane fraction thereof according to (30) and (ii) galanin and the test compound are contacted with the cell or the cell membrane fraction thereof according to (30);
(96) a pharmaceutical composition according to (45) for inhibiting liberation of acetylcholine, inhibiting secretion of insulin, stimulating secretion of growth hormones, inhibiting learning behavior or inhibiting satiety;
(97) a pharmaceutical composition according to (45) which is a prophylactic or therapeutic agent for schizophrenic illness or stomach ulcer or is a sedative;
(98) a pharmaceutical composition according to (48) for promoting the acetylcholine liberation, inhibiting the growth hormone secretion, promoting the insulin secretion, promoting the learning behavior or promoting satiety;
(99) a pharmaceutical composition according to (48) which is a prophylactic and therapeutic agent for diabetes, Alzheimer""s disease or dementia;
(100) a preventive and therapeutic agent containing the DNA according to (18) for a galanin receptor protein-deficient disease; and
(101) a preventive and therapeutic agent according to (100) in which the galanin receptor protein-deficient disease is diabetes, Alzheimer""s disease or dementia.
Yet another aspect of the present invention is:
(102) a galanin receptor protein according to the above (1) which comprises
an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 1, amino acid sequences wherein one or more amino acid residues (preferably from 1 to 30 amino acid residues, more preferably from 1 to 10 amino acid residues) are deleted from the amino acid sequence of SEQ ID NO: 1, amino acid sequences wherein one or more amino acid residues (preferably from 1 to 30 amino acid residues, more preferably from 1 to 10 amino acid residues) are added to the amino acid sequence of SEQ ID NO: 1, and amino acid sequences wherein one or more amino acid residues (preferably from 1 to 30 amino acid residues, more preferably from 1 to 10 amino acid residues) in the amino acid sequence of SEQ ID NO: 1 are substituted with one or more other amino acid residues,
or a salt thereof;
(103) a galanin receptor protein according to the above (1) which comprises
an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 2, amino acid sequences wherein one or more amino acid residues (preferably from 1 to 30 amino acid residues, more preferably from 1 to 10 amino acid residues) are deleted from the amino acid sequence of SEQ ID NO: 2, amino acid sequences wherein one or more amino acid residues (preferably from 1 to 30 amino acid residues, more preferably from 1 to 10 amino acid residues) are added to the amino acid sequence of SEQ ID NO: 2, and amino acid sequences wherein one or more amino acid residues (preferably from 1 to 30 amino acid residues, more preferably from 1 to 10 amino acid residues) in the amino acid sequence of SEQ ID NO: 2 are substituted with one or more other amino acid residues,
or a salt thereof; and
(104) a galanin receptor protein according to the above (1) which comprises an amino acid sequence selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 5, amino acid sequences wherein one or more amino acid residues (preferably from 1 to 30 amino acid residues, more preferably from 1 to 10 amino acid residues) are deleted from the amino acid sequence of SEQ ID NO: 5, amino acid sequences wherein one or more amino acid residues (preferably from 1 to 30 amino acid residues, more preferably from 1 to 10 amino acid residues) are added to the amino acid sequence of SEQ ID NO: 5, and amino acid sequences wherein one or more amino acid residues (preferably from 1 to 30 amino acid residues, more preferably from 1 to 10 amino acid residues) in the amino acid sequence of SEQ ID NO: 5 are substituted with one or more other amino acid residues,
or a salt thereof.
Yet another aspect of the present invention is:
(105) a process according to the above (27), wherein said transformant is produced by transforming a host cell, CHO cell, with a vector comprising a nucleotide sequence coding for a human-derived galanin receptor protein;
(106) a pharmaceutical composition comprising an effective amount of an agonist according to the above (40) or a salt thereof in admixture with a pharmaceutically acceptable diluent, carrier or excipient;
(107) a pharmaceutical composition according to the above (106), which inhibits liberation of acetylcholine, secretion insulin, learning action, or satiety;
(108) a pharmaceutical composition comprising an effective amount of an antagonist according to the above (40) or a salt thereof in admixture with a pharmaceutically acceptable diluent, carrier or excipient;
(109) a pharmaceutical composition according to the above (108), which promotes liberation of acetylcholine, secretion insulin, learning action, or satiety; and
(110) a transformant CHO cell capable of expressing human-derived galanin receptor proteins.
As used herein the term xe2x80x9csubstantial equivalent(s)xe2x80x9d means that the activity of the protein, e.g., nature of the ligand binding activity, and physical characteristics are substantially the same. Substitutions, deletions or insertions of amino acids often do not produce radical changes in the physical and chemical characteristics of a polypeptide, in which case polypeptides containing the substitution, deletion, or insertion would be considered to be substantially equivalent to polypeptides lacking the substitution, deletion, or insertion. Substantially equivalent substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. The non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.